**7. GSIS amplification by incretins GLP-1 and GIP**

Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) have a prominent impact among other peptides belonging to incretins [107–109]. Oral glucose administration provides a higher insulin secretion response than when administered parenterally [146]. This surplus of potentiation of insulin secretion appears to be about equally ascribed to GLP-1 and GIP [147]. Indeed, diminished insulin secretion response to oral glucose was observed in GLP-1 knock out mice [148, 149] and was even more decreased in double knockout mice (GLP-1 plus GIP) [149].

Incretin-cAMP signaling amplifies GSIS by both PKA-dependent and EPAC2Adependent pathways. As described above, the EPAC2 pathway is partially dependent on the CaL opening, and the PKA pathway enables synergy among actions of KATP, CaL, and Kv channels, leading again to a more effective CaL opening. This knowledge complies with the traditional view, considering that the incretin signaling does not stimulate insulin release in the low glucose conditions [150, 151]. The GLP1RcAMP-EPAC2-TRPM2 pathway was suggested to be one of the major routes [106].

GLP-1 is secreted by enteroendocrine L-cells, residing predominantly in the distal ileum and colon. Secretion is initiated by postprandial stimuli, i.e. by glucose,

**43**

**keto-acids**

*Redox Signaling is Essential for Insulin Secretion DOI: http://dx.doi.org/10.5772/intechopen.94312*

beyond the scope of this review.

found even for the internalized GLP1R [164].

fatty acids, or lipids, as well as proteins [152, 153]. Only 10 to 15% of active GLP-1 likely reaches the pancreas via the circulation [154]. Thus concentrations of biologically active GLP-1 in human plasma at fasting account for about 2 pmol/l and maximum10 pmol/l postprandially [155], peaking 30 to 60 min after a carbohydrate or protein intake and 120 min after ingestion of lipids [156]. The most efficient truncated variants are GLP-1(7–37) and variant GLP-1(7–36amide) [152]. The latter is ~80% abundant in humans [157]. Note that full peptide GLP-1(1–37) is much less efficient in GSIS potentiation [150, 151]. Moreover, paracrine GLP-1 signaling acts among the different types of PI cells [150], similarly to the paracrine and endocrine secretion of other hormones. On the systemic level, central control by the brain and nervous system, including GLP-1 secretion in the *nucleus tractus solitarii* of the brainstem [152], further provides an indispensable top level of regulation for the insulin secretion. GLP-1 effects related to β-cell proliferation or apoptosis are

GLP-1 from the bloodstream acts through its receptor (GLP1R) residing in the plasma membrane of pancreatic β-cells [158]. GLP1R activation stimulates Gαs and Gαq/11 and recruits β-arrestin, depending on biased agonism relative to different agonists, such as exendin-4 and oxyntomodulin [159, 160]. As a scaffold protein, β-arrestin facilitates signaling via Gαs to cAMP but also to CREB [160], extracellular regulated kinase ERK1/2 [161], and insulin receptor substrate 2 (IRS-2), the effects promoting β-cell growth, differentiation, and maintenance [160]. The stimulation of Gαs leads via enhanced cAMP to the initiation of PKA [162] and EPAC2A pathways [163]. Continuous cAMP production and partial potentiation of GSIS was

The PKA pathway provides a surplus intracellular Ca2+ above that of the net GSIS without any receptor stimulation. This is ensured by phosphorylation-induced closing among the population of KATP, stimulation of CaL opening, and closing of Kv channels [165]. The latter prolongs Ca2+ stimulation of IGV exocytosis and hence may also potentiate the 2nd phase of GSIS. In parallel, PKA engages snapin interaction with IGVs, reportedly potentiating the 1st GSIS phase [123, 124]. Simultaneously, the EPAC2 pathway promotes Ca2+-induced RyR-mediated Ca2+ release from ER, which must be, however, initiated by the ongoing CaL opening [163]. The EPAC2 pathway also facilitates docking and priming of IGVs by promoting Rab3A interaction with Rim2a [131] and hypothetically interaction of EPAC2- Rim2-Picollo trimers with Rab3A, enabling IGV exocytosis [152]. Stimulation of GLP1R biased downstream via stimulation of Gαq/11 also contributes by a surplus to intracellular Ca2+, while inducing the IP3R-mediated Ca2+ release from ER. When GLP1 effects were simulated and IGV kinetics was monitored using total internal reflection fluorescence microscopy, cAMP and 8-Br-cAMP were found to increase the frequency of fusion events, i.e. IGV fusion with the plasma membrane in both phases of GSIS [25]. EPAC2A was found to interacts also with a small G protein Rap1, affecting its conformation so to release the catalytic region, which subsequently binds and thus activates another G protein Rap113. In EPAC2A knockout mice, most of the potentiation of the 1st GSIS phase vanished [25]. Thus

speculatively, the 2nd phase amplification can be due to the PKA pathway.

**8. Mechanism of insulin secretion stimulated by branched-chain** 

Postprandial response by insulin secretion is also given by substances other than glucose. These substances, which induce the secretion of insulin, are termed secretagogues in general. One important type of secretagogues is branched-chain *Type 2 Diabetes - From Pathophysiology to Cyber Systems*

hence mitochondrial ATP synthesis [145].

**7. GSIS amplification by incretins GLP-1 and GIP**

Glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) have a prominent impact among other peptides belonging to incretins [107–109]. Oral glucose administration provides a higher insulin secretion response than when administered parenterally [146]. This surplus of potentiation of insulin secretion appears to be about equally ascribed to GLP-1 and GIP [147]. Indeed, diminished insulin secretion response to oral glucose was observed in GLP-1 knock out mice [148, 149] and was even more decreased in double knockout mice (GLP-1 plus GIP) [149]. Incretin-cAMP signaling amplifies GSIS by both PKA-dependent and EPAC2Adependent pathways. As described above, the EPAC2 pathway is partially dependent on the CaL opening, and the PKA pathway enables synergy among actions of KATP, CaL, and Kv channels, leading again to a more effective CaL opening. This knowledge complies with the traditional view, considering that the incretin signaling does not stimulate insulin release in the low glucose conditions [150, 151]. The GLP1RcAMP-EPAC2-TRPM2 pathway was suggested to be one of the major routes [106]. GLP-1 is secreted by enteroendocrine L-cells, residing predominantly in the distal ileum and colon. Secretion is initiated by postprandial stimuli, i.e. by glucose,

then interacts with Rab3A and enables IGV exocytosis.

The local Ca2+ influx within CaL ensures EPAC2 binding to Rim2, and subsequent interaction with another Ca2+ sensor termed Piccolo. The heterotrimeric complex

Interestingly, all necessary components of the PKA pathway were identified in the mitochondrial matrix, including sAC, PDE2A2 [132], and also PKA [133]. However, we may also speculate that some proteins can be phosphorylated by cytosolic PKA or by its fraction attached to OMM prior to their import to the mitochondrial matrix. There was also a consensus that cAMP cannot freely diffuse to the matrix [132]. Thus cAMP in the mitochondrial matrix may act as an independent pool [134, 135]. Its source is the matrix-located soluble adenylate cyclase sAC, which is activated by bicarbonate and Ca2+ [136, 137]. Since CO2 is increasingly released when the Krebs cycle turnover increases upon GSIS, the matrix localized mtPKA can be activated in this way [138]. In any case, OXPHOS is facilitated in mitochondria of numerous tissues via phosphorylation of Complex I NDUFS4 subunit (facilitating its Hsp70-mediated import), Complex IV COXIV-1 subunit (preventing its inhibition by ATP) [139] as well as via IF1, enhancing ATP synthesis by disabling the inhibitory binding of phosphorylated IF1 dimers to the ATP synthase [140]. A link to redox homeostasis can be viewed in the observed release of the PKA catalytic subunits by the increased ROS [141, 142]. Thus mtPKA can act in parallel to the cytosolic PKA signaling initiated by GPR40 and GLPR or GIPR receptors. PKA targeting of at least IF1, and probably also of Complex I and Complex IV, should contribute to the amplification of insulin secretion by FAs or incretins. The G protein Gαq/11 initiates signaling through the phospholipase C (PLC-) mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate into DAG and inositol triphosphate IP3 [110]. The main effector of DAG is protein kinase C (PKC), which is activated by DAG. One of the effectors of IP3 is the IP3 receptor (IP3R; subtypes IP3R1, IP3R2 and IP3R3), which is another important Ca2+ channel residing on ER membranes in β-cells [143]. Similarly to the EPAC2-RyR route of Ca2+ release from ER Ca2+, the opening of this channel amplifies the primary CaL mediated Ca2+ signaling for insulin release. PKC contributes to the plasma membrane depolarization, while activating TRPM4 and TRPM5 [144]. Besides the canonical plasma membrane effects, PKC and downstream ERK1/2 signaling stimulates OXPHOS,

**42**

fatty acids, or lipids, as well as proteins [152, 153]. Only 10 to 15% of active GLP-1 likely reaches the pancreas via the circulation [154]. Thus concentrations of biologically active GLP-1 in human plasma at fasting account for about 2 pmol/l and maximum10 pmol/l postprandially [155], peaking 30 to 60 min after a carbohydrate or protein intake and 120 min after ingestion of lipids [156]. The most efficient truncated variants are GLP-1(7–37) and variant GLP-1(7–36amide) [152]. The latter is ~80% abundant in humans [157]. Note that full peptide GLP-1(1–37) is much less efficient in GSIS potentiation [150, 151]. Moreover, paracrine GLP-1 signaling acts among the different types of PI cells [150], similarly to the paracrine and endocrine secretion of other hormones. On the systemic level, central control by the brain and nervous system, including GLP-1 secretion in the *nucleus tractus solitarii* of the brainstem [152], further provides an indispensable top level of regulation for the insulin secretion. GLP-1 effects related to β-cell proliferation or apoptosis are beyond the scope of this review.

GLP-1 from the bloodstream acts through its receptor (GLP1R) residing in the plasma membrane of pancreatic β-cells [158]. GLP1R activation stimulates Gαs and Gαq/11 and recruits β-arrestin, depending on biased agonism relative to different agonists, such as exendin-4 and oxyntomodulin [159, 160]. As a scaffold protein, β-arrestin facilitates signaling via Gαs to cAMP but also to CREB [160], extracellular regulated kinase ERK1/2 [161], and insulin receptor substrate 2 (IRS-2), the effects promoting β-cell growth, differentiation, and maintenance [160]. The stimulation of Gαs leads via enhanced cAMP to the initiation of PKA [162] and EPAC2A pathways [163]. Continuous cAMP production and partial potentiation of GSIS was found even for the internalized GLP1R [164].

The PKA pathway provides a surplus intracellular Ca2+ above that of the net GSIS without any receptor stimulation. This is ensured by phosphorylation-induced closing among the population of KATP, stimulation of CaL opening, and closing of Kv channels [165]. The latter prolongs Ca2+ stimulation of IGV exocytosis and hence may also potentiate the 2nd phase of GSIS. In parallel, PKA engages snapin interaction with IGVs, reportedly potentiating the 1st GSIS phase [123, 124]. Simultaneously, the EPAC2 pathway promotes Ca2+-induced RyR-mediated Ca2+ release from ER, which must be, however, initiated by the ongoing CaL opening [163]. The EPAC2 pathway also facilitates docking and priming of IGVs by promoting Rab3A interaction with Rim2a [131] and hypothetically interaction of EPAC2- Rim2-Picollo trimers with Rab3A, enabling IGV exocytosis [152]. Stimulation of GLP1R biased downstream via stimulation of Gαq/11 also contributes by a surplus to intracellular Ca2+, while inducing the IP3R-mediated Ca2+ release from ER.

When GLP1 effects were simulated and IGV kinetics was monitored using total internal reflection fluorescence microscopy, cAMP and 8-Br-cAMP were found to increase the frequency of fusion events, i.e. IGV fusion with the plasma membrane in both phases of GSIS [25]. EPAC2A was found to interacts also with a small G protein Rap1, affecting its conformation so to release the catalytic region, which subsequently binds and thus activates another G protein Rap113. In EPAC2A knockout mice, most of the potentiation of the 1st GSIS phase vanished [25]. Thus speculatively, the 2nd phase amplification can be due to the PKA pathway.
